Production Of Beta 2-microglobulin Research Articles

Quantitative determination of the beta-2-microglobulin monomer by gel filtration chromatography

Beta-2-microglobulin (B2M) is an important material in dialysis-related amyloidosis research. Unfortunately, the quantitative detection of the B2M monomer is difficult during B2M production. In this study, we established a method for the detection of the B2M monomer and its dimer in solution via gel filtration chromatography. The B2M powder was dissolved in 0.01 mol/L phosphate-buffered solution (PBS, pH 7.2-7.4) with a final mass concentration of 0.5 g/L. The B2M sample was analyzed on a TSKgel SuperSW2000 column (30 cm×4.6 mm, 4 μm) by an Agilent 1200 Series HPLC using 0.01 mol/L PBS (pH 7.2-7.4) as the mobile phase at a flow rate of 0.5 mL/min. The temperature of the column was 25℃, and the detection wavelength was 280 nm. The purities of the B2M monomer and the B2M dimer were tested. A series of concentrations of B2M monomer solutions were prepared to create a standard working curve. The standard working curve of the B2M monomer had a good linear relationship (coefficient of correlation (r2) was 0.9948). The limit of quantitation for the B2M monomer in PBS was 0.08 g/L (S/N=10). The recoveries were 85.0%-96.7% with relative standard deviations of 1.7%-3.3% at spiked levels of 0.10-0.30 g/L. The quantification of the B2M monomer was undisturbed by the B2M dimer, which can form during the B2M purification process. This determination method is simple, stable, and reliable for the determination of the B2M monomer in B2M industrial production.

Dissociation between beta-2 microglobulin and IL-1 production in hemodialyzed patients.

beta-2 microglobulin is predominant in amyloid deposits in patients undergoing long term hemodialysis. Amyloid accumulation has been ascribed to dialysis membranes, endotoxin contamination of the dialysate, uremia and chronic systemic inflammation associated with enhanced monocytic cytokine production in hemodialyzed patients. Interleukin-1 has been proposed to play a critical role in the induction of beta-2 microglobulin synthesis and release. We examined if monocytes contribute to beta-2 microglobulin production upon stimulation with inflammatory mediators that are generated during hemodialysis and investigated the production of beta-2 microglobulin by cells from patients, with and without clinical signs of amyloidosis, at the time when patients' monocytes contained maximal intracellular accumulation of IL-1. We demonstrated that only monocytes are able to release increased levels of beta-2 microglobulin upon stimulation by IL-1, TNF alpha, C5a and LPS. Increased levels of beta-2 microglobulin were associated with increased levels of beta-2 microglobulin mRNA. Before dialysis session, 20-60% of circulating CD14+ monocytes from patients contained IL-1. At the time when maximal IL-1 production was detected, we showed by RT-PCR increased transcription of IL-1 gene in patients' monocytes. We observed that monocytes from patients with amyloidosis contained higher amounts of IL-1 as compared to monocytes from patients without clinical signs of amyloidosis, but could not secrete increased amounts of beta-2 microglobulin upon LPS-stimulation. Our data indicated that chronic inflammation, as demonstrated by increased intracellular IL-1 expression, is not associated with increased production of beta-2 microglobulin by monocytes from patients on hemodialysis.

Comparison of spinal fluid beta 2-microglobulin levels with CD4+ T cell count, in vitro T helper cell function, and spinal fluid IgG parameters in 163 neurologically normal adults infected with the human immunodeficiency virus type 1.

Beta 2-microglobulin levels were measured in the cerebrospinal fluid (CSF) and serum of 163 human immunodeficiency virus-positive (HIV+) persons with normal neurologic physical examinations. None were on antiretroviral therapy. Only 3% had a positive CSF HIV p24 antigen test. The CSF beta 2-microglobulin levels increased as the CD4+ T cell count decreased. Intrathecal production of beta 2-microglobulin was suggested by finding CSF concentrations greater than serum concentrations in 15% of patients. The CSF beta 2-microglobulin levels rose as in vitro T helper cell function deteriorated, independent of CD4+ T cell count. CSF beta 2-microglobulin levels paralleled CSF IgG, IgG index, and IgG synthesis. Higher CSF beta 2-microglobulin levels were found in persons with positive CSF oligoclonal bands. CSF beta 2-microglobulin concentration may serve as a marker for subclinical neurologic damage due to HIV. If this is established, defining the effect of anti-HIV interventions on CSF beta 2-microglobulin would be warranted.

Increased serum beta 2-microglobulin concentrations in hyperthyroid states.

Serum beta 2-microglobulin concentrations were determined in 21 untreated hyperthyroid patients (12 with Graves' disease, and nine with toxic nodular adenoma) and in 20 healthy controls. All subjects had normal serum creatinine concentrations and urine analysis. Both total and free thyroid hormones were significantly higher in the hyperthyroid groups than in controls. Beta 2-microglobulin concentrations were significantly increased in both groups of hyperthyroid patients compared with controls. No difference was found in the thyroid hormone and beta 2-microglobulin concentrations between both sets of patients. The beta 2-microglobulin and thyroid hormone concentrations were not correlated. These data show that hyperthyroidism is another cause of increased beta 2-microglobulin production along with viral infections, immunologically mediated diseases, and malignant neoplasms. The increased serum beta 2-microglobulin concentration in thyroid hyperfunction is probably related to metabolic rate, even if autoimmunity might contribute to its overproduction.

Effects of dialysis membranes on beta2-mieroglobulin production and cellular expression

We investigated the effects of different dialysis membranes on the production of beta 2-microglobulin (beta 2m) in peripheral blood mononuclear cell cultures (PBMNC) obtained from hemodialysis patients in a prospective cross-over design study. Chronic dialysis with cuprophane membrane leads to an increase in beta 2m production from 129 +/- 11 ng/ml to 192 +/- 23 ng/ml (P less than 0.002). This increase is reversed by the use of a non-complement activating membrane polymethylmethacrylate. In addition, during chronic dialysis with cuprophane membrane, an increasing proportion of these cells display low beta 2m expression on their surface (from 6.1 +/- 0.8% of PBMNC to 16.9 +/- 3.4%, P less than 0.001), concomitant with the emergence of cells with low density of HLA on their surface (from 4.9 +/- 1.2% of cells to 32.9 +/- 7.8% of cells, P less than 0.001). The total content of cell-associated beta 2m is also decreased in dialysis patients in general, and in particular in patients chronically dialyzed with new cuprophane membrane. These effects can be reproduced by incubation of PBMNC with cuprophane membranes, and with the addition of C5a, IL-1 and TNF in vitro. Thus, chronic dialysis with cuprophane membrane may be a factor in the genesis of high beta 2m levels and causes changes in beta 2m and HLA expression on cell surfaces.

Metabolic and renal changes in two athletes during a world 24 hour relay record performance.

Metabolic parameters and renal function were studied in two subjects before, during and after they established a world two-man 24 hour relay record. During the race, the athletes expended an estimated 37.747 and 42.880 kJ running at 54 and 61 per cent of maximum oxygen consumption (VO2max). Rectal temperatures reached maxima of 38.6 and 39.2 degrees C respectively during the race. Serum free fatty acid levels peaked at 2108 and 1875 mumol ml-1 after 24 hours; blood glucose levels varied from 4.3-6.5 and 4.9-8.5 mmol.l-1 respectively. Plasma insulin levels fell from 42.9 and 22.7 microU.ml-1 to 11.5 microU.ml-1. Plasma urea, creatinine, beta 2-microglobulin and C-reactive protein concentrations were elevated at the end of the race (to 9.0 and 8.0 mmol.l-1, 119 and 102 mumol.l-1, 3.508 and 3203 micrograms.l-1 and 2.7 and 3.9 mg per cent respectively). Plasma osmolality was altered from 293 and 304 to 302 and 280 mosmol.Kg-1 during the race but increased to 312 and 318 mosmol.Kg-1 the following day probably due to intercompartmental fluid shifts. Plasma creatinine concentration was increased by 38 and 26 per cent due to reduced urinary excretion. Urine flow rate increased 40 and 123 per cent respectively during the race, but creatinine clearance decreased by 38 and 40 per cent. Urine osmolality decreased by 38 and 65 per cent and osmolal clearance decreased by 15 and 16 per cent respectively. Urine sodium excretion was greatly reduced (85 and 90 per cent) on the post-race days (by 88 and 92 per cent on day 2). Both urine total protein and beta2-microglobulin excretion increased during the race (by 89 and 35 per cent and by 334 and 136 per cent respectively), but owing to the increased beta2-microglobulin production renal clearance was unaltered. The changes in renal function were temporary and some aspects of renal tubular function were enhanced during the post-race days. We conclude that, although C-reactive protein concentrations increased sooner and were higher than other shorter events and although creatinine, urine excretion and urine osmolality decreased markedly, the intermittent nature of the event, the mild environmental conditions, the moderate percentage of VO2 max maintained by the well conditioned subjects and a high fluid intake enabled a rapid return to normality and indeed to enhanced renal tubular function. The only moderate increases in body temperature would be due to the same factors.

Comparison of serum and synovial fluid concentrations of beta 2-microglobulin and C reactive protein in relation to clinical disease activity and synovial inflammation in rheumatoid arthritis.

beta 2-Microglobulin and C reactive protein (CPR) were measured in 33 and 57 matched pairs of serum and synovial fluid (SF) respectively, from patients with active rheumatoid arthritis (RA). Serum beta 2-microglobulin concentrations were higher than in normal controls and the SF concentration was higher than the serum concentration on 25 of 33 occasions (76%), suggesting a local production of beta 2-microglobulin within the synovial membrane. There was a correlation between serum and SF concentrations of beta 2-microglobulin (r = 0.50). Patients' serum CRP concentrations in 57 samples were higher than in normal controls and were greater than in the matched SFs on 49 of the 57 paired samples (86%). In 18 samples CRP was absent in the SF, suggesting a local consumption or binding within the synovial membrane. Twenty four patients with RA given either sodium aurothiomalate or D-penicillamine for six months showed highly significant clinical improvements accompanied by reductions in serum and SF immunoglobulin concentrations and knee joint suprapatellar pouch synovial membrane T lymphocyte infiltrates. In this group of patients serum CRP, but not beta 2-microglobulin, fell significantly, but there were no significant changes in SF beta 2-microglobulin or CRP. These data suggest that serum and SF beta 2-microglobulin concentrations are not a useful index for determining the therapeutic response to sodium aurothiomalate and D-penicillamine and that serum rather than SF CRP concentrations are more helpful. The persistent raised serum and SF concentrations of beta 2-microglobulin probably reflect synovial inflammatory infiltrates, which are still considerable despite apparent clinical remission.

Clearance of beta-2-microglobulin using continuous ambulatory peritoneal dialysis.

In the 9 continuous ambulatory peritoneal dialysis (CAPD) patients studied, the mean clearance of beta 2-microglobulin was significantly higher using hypertonic as opposed to isotonic exchanges (1 ml/min and 0.75 ml/min, respectively). Clearance of beta 2-microglobulin correlated with the clearance of albumin. The daily mass transfer of beta 2-microglobulin ranged from 19 to 62 mg. Although all the daily production of beta 2-microglobulin is not eliminated in patients on CAPD their serum beta 2-microglobulin levels would be expected to be lower than in patients on haemodialysis. Long-term prospective studies are needed to determine whether lower serum beta 2-microglobulin levels lead to a lower incidence of dialysis amyloid.

Alpha‐ and gamma‐interferon (IFNα, IFNγ) but not interleukin‐1 (IL‐1) modulate synthesis and secretion of β2‐microglobulin by hepatocytes

Soluble serum beta 2-microglobulin has been thought to result from membrane shedding by activated T-lymphocytes. This hypothesis could explain the increase of beta 2-microglobulin serum levels during virally induced mononucleosis, but not elevated levels as observed in other virally induced and in malignant diseases. In this paper we demonstrate that beta 2-microglobulin is a true secretory protein, and that its synthesis in hepatocytes is modulated by IFNs but not by IL-1. While the 45,000 MW HLA antigen can be found only in cell lysates, beta 2-microglobulin is shown to be secreted also into the culture medium like other secretory proteins (e.g. albumin-factor B-complement C3). Furthermore, interferon alpha (IFN alpha) as well as interferon gamma (IFN gamma) directly stimulate, in a dose- and time-dependent manner, beta 2-microglobulin synthesis by human hepatoma cells (Mz-Hep-1 and PLC/PRF5) and murine hepatocyte primary cultures. The increase of beta 2-microglobulin production induced by interferons is demonstrated at both the protein and the RNA level, indicating that interferon acts at a pretranslational level. The interferon effect on beta 2-microglobulin synthesis is specific since synthesis of secretory proteins like complement C3 or albumin, and of a structural protein like actin, remains unchanged. In contrast to IFN, IL-1, the main mediator of acute phase response, does not change beta 2-M biosynthesis rate. These data indicate that (i) beta 2-microglobulin is a secretory protein, (ii) IFNs but not IL-1 can mediate increased beta 2-M serum levels, and (iii) the liver may be its primary source.

Production of beta 2-microglobulin by chronic lymphocytic leukaemia cells in vitro.

The in vitro production of beta 2-microglobulin (beta 2m) by leukaemic cells was studied in 22 patients with chronic B-lymphocytic leukaemia (CLL). In addition, the concentration of beta 2m in serum (S-beta 2m) was determined and expressed as percent of the upper normal limit, after a correction for elevated S-Creatinine values. Patients with progressive disease usually had CLL cells with a high rate of in vitro synthesis and an increased S-beta 2m. This was not found in patients with non-progressive disease. The in vitro synthesis of beta 2m X the lymphocyte count correlated with S-beta 2m in the total material (r = 0.65). The increased S-beta 2m frequently observed in CLL may therefore originate from the tumour cells. Hence, S-beta 2m is promising as a clinically useful tumour cell-associated marker in CLL.

Beta 2-Microglobulin secretion from lymphoid cells: a study at the cellular level.

A protein plaque assay was developed for the detection of human lymphocytes secreting beta 2-microglobulin. The development of plaques required production of beta 2-microglobulin by viable cells at an estimated rate exceeding 25 molecules per second. It has previously been suggested that beta 2-microglobulin secretion is restricted to certain lymphoid cell subpopulations. However, both T- and B-cells were shown to form plaques in normal donors and immunodeficient patients after activation with various mitogens. A close correlation between beta 2-microglobulin production and DNA synthesis was evident. However, secretion of immunoglobulin did not correlate with the proportion of cells secreting beta 2-microglobulin.

Beta 2-microglobulin concentration in plasma and production in liver cirrhosis.

The beta 2-microglobulin plasma level is often high in patients suffering from cirrhosis. Many authors believe this to be due to an increased production, provided that the creatinine level is in the normal range. In the present study, alterations in the plasma level and production of beta 2-microglobulin were investigated in patients with liver cirrhosis without overt renal failure. 62 patients, 48 men and 14 women, suffering from liver cirrhosis were examined. The glomerular filtration rate (GFR) and plasma beta 2-microglobulin were measured in all patients and in 16 controls. As beta 2-microglobulin is freely filtered by glomeruli and its extrarenal catabolism is negligible, the beta 2-microglobulin filtration rate was calculated as the product of the beta 2-microglobulin plasma level times the GFR. In steady state conditions, the beta 2-microglobulin filtration rate may be used as an indirect index of beta 2-microglobulin production. The beta 2-microglobulin plasma level was high in 26 patients; however, only 12 of them showed a definite rise in beta 2-microglobulin production, as shown by an increased beta 2-microglobulin filtration rate. The 14 patients with high beta 2-microglobulin plasma levels without high beta 2-microglobulin filtration rates obviously showed a decreased GFR; however, creatinine was not increased because of its small sensitivity as an index of renal function. A linear correlation was found between IgG and the beta 2-microglobulin filtration rate (r = 052; p less than 0.02), not between IgG and the beta 2-microglobulin plasma level. The other indices of liver damage were not related to the beta 2-microglobulin filtration rate of plasma level.

Human Malignant Lymphomas in Vitro

A series of 55 biopsies from different types of malignant lymphomas were characterized in short-term culture experiments and during prolonged growth in vitro. The majority of the lymphocytic lymphomas and half of the histiocytic lymphomas expressed surface immunoglobulin, either in monoclonal or polyclonal form, indicating B-lymphocyte derivation. No lysozyme production was noted in either type of lymphoma, giving further support to the notion that histiocytic lymphomas are not truly histiocytic. Production of beta2-microglobulin was higher in histiocytic than in lymphocytic lymphoma and Hodgkin's disease but did not significantly differ from the production observed in non-neoplastic lymph node disorders. Incorporation of 3H-thymidine varied greatly within each category of lymphoma; the highest mean labelling index was noted in histiocytic lymphoma, possibly reflecting the generally more malignant course in such cases. Epstein-Barr virus-associated nuclear antigen was observed in one case of Hodgkin's disease. Attempts to establish permanent tumor cell lines were successful only from two explants of lymphocytic lymphoma and one pleural effusion from histiocytic lymphoma. The two cell lines derived from lymphocytic lymphomas both exhibited B-lymphocyte characteristics. The histiocytic lymphoma line lacked lymphocyte markers, produced lysozyme and was found to be rich in cytoplasmic esterases. These features are consistent with a "true" histiocytic derivation of this line. Lymphoblastoid cell lines representing non-neoplastic EBV-carrying lymphocytes contaminating the biopsies were derived from 19 biopsies, with the highest frequency noted in cultures of biopsies from Hodgkin's disease. The tumor lines were all EBV-genome negative.

Structural abnormalities in chromosome 15 in cell lines with reduced expression of beta-2 microglobulin

Using somatic cell hybrids, the gene for beta-2 microglobulin has been assigned to human chromosome 15. We found it of interest to study a number of human lymphoid cell lines in light of this finding, to analyze whether spontaneously occurring loss or reduction of beta-2 microglobulin could be correlated with any aberration in chromosome 15. The Daudi cell line was shown to be devoid of any beta-2 microglobulin in total extracts. Chromosome analysis showed that one of the two chromosomes 15 was deleted in the region q14↔q21 on the long arm; in some metaphases, both chromosomes were deleted in this region. The K562 cell line was found to express very low (if any) membrane-associated beta-2 microglobulin. Chromosome analysis showed that this line was near-triploid, with two normal chromosomes 15 and one translocation chromosome t(15;18) involving the long arm of chromosome 15, whereby the segment proximal to the breakage point in band q15 was lost. The Namalwa cell line showed a reduction in membrane-associated and total beta-2 microglobulin. Chromosome analysis showed this line to contain one chromosome 15 which was shorter than its normal homolog. The deletion could be identified as such in the region q14↔q21 in Daudi cells, but is probably somewhat smaller than the one in Daudi cells. Since analyses of beta-2 microglobulin production and chromosomes 15 on several other human cells failed to reveal any abnormalities in either of these respects, we postulate that genes responsible for beta-2 microglobulin synthesis and membrane expression could be located in the region q14→q21 on the long arm of chromosome 15. Since beta-2 microglobulin associated with the membrane was found to be absent in the K562 line, where total beta-2 microglobulin was nearly as high as in cell lines with “normal” membrane expression, it is suggested that membrane expression of beta-2 microglobulin can be regulated independently

Establishment and characterization of a human histiocytic lymphoma cell line (U‐937)

A human hematopoietic cell line (U-937) with exceptional characteristics was derived from a patient with generalized histiocytic lymphoma. The morphology of the cell line was identical to that of the tumor cells in the pleural effusion from which the line was derived. Since Epstein-Barr virus (EBV) carrying diploid lymphoblastoid cell lines unrelated to the tumor population often become established in vitro from non-Burkitt lymphoma explants, several parameters were studied to discriminate the U-937 from such lines: morphology in vitro, growth characteristics, cytochemistry, surface receptor pattern, Ig production, lysozyme production, beta2-microglobulin production, presence of EBV genome and karyotype. In all these respects U-937 differed from prototype lymphoblastoid cell lines. The histiocytic origin of the cell line was shown by its capacity for lysozyme production and the strong esterase activity (naphtol AS-D acetate esterase inhibited by NaF) of the cells. It is therefore concluded that the U-937 is a neoplastic, histiocytic cell line.

Production of beta 2-microglobulin by normal and malignant human cell lines and peripheral lymphocytes.

Production of beta 2-microglobulin by normal and malignant human cell lines and peripheral lymphocytes.